WO2014047199A1 - Nouveaux promédicaments pour une thérapie anticancéreuse sélective - Google Patents

Nouveaux promédicaments pour une thérapie anticancéreuse sélective Download PDF

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WO2014047199A1
WO2014047199A1 PCT/US2013/060443 US2013060443W WO2014047199A1 WO 2014047199 A1 WO2014047199 A1 WO 2014047199A1 US 2013060443 W US2013060443 W US 2013060443W WO 2014047199 A1 WO2014047199 A1 WO 2014047199A1
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compound
linker
oligopeptide
amino acid
aryl
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PCT/US2013/060443
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Nobuhide Ueki
Michael J. HAYMAN
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The Research Foundation For The State University Of New York
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Publication of WO2014047199A1 publication Critical patent/WO2014047199A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
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    • A61K31/00—Medicinal preparations containing organic active ingredients
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    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/067—Pyrimidine radicals with ribosyl as the saccharide radical
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/09—Pyrimidine radicals with arabinosyl as the saccharide radical
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    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
    • C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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    • C07K5/08—Tripeptides
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    • C07K5/10—Tetrapeptides
    • C07K5/1019—Tetrapeptides with the first amino acid being basic

Definitions

  • a therapeutic agent or drug having low selectivity leads to reduced efficacy and higher toxicity.
  • a ma or limitation of many cancers treatments is their low selectivity for tumor cells.
  • Radiation therapy and alkylating agents perturb many functions that are common to both tumor and normal cells .
  • HDACs are key enzymes involved in the epigenetic regulation of histone and non-histone proteins (Witt, 0. et al . 2009) . They modulate protein structure and function through deacetylation of lysine residues. In cancer biology, the involvement of HDACs has been well documented, supporting the notion that altered expression of HDACs could have an active role in tumor development (Haberland, M. et al. 2009; Bolden, J.E. et al . 2006) . Consistent with this, the therapeutic potential of HDAC inhibitors (HDACi) is recognized as a new class of drug for cancer (Bolden, J.E. et al . 2006; Minucci, S. et al. 2006; Marks, P. A.
  • HDAC inhibitors which were developed as single target agents, are a new class of drugs for cancer (Minucci, S. & Pelicci, P.G. 2006; Bolden, J.E. et al. 2006; Marks, P. A. & Xu, W.S. 2009).
  • HDACi HDAC inhibitors
  • several HDACi have been found to have potent anticancer effects.
  • Tumor-associated cysteine protease CTSL also plays crucial roles at multiple stages of tumor progression and metastasis (Joyce, J. . et al . 2004; Luciszko, C. et al. 2004; Gonzalez-Suarez , I. et al . 2011) .
  • Cell lines transformed by certain oncogenes including Ras are known to express high levels of CTSL (Collette, J. et al . 2004; Denhardt, D.T. et al . 1987; Joseph, L.J. et al . 1988).
  • the upregulation of CTSL is recognized as a hallmark of metastatic cancers and could be utilized as a prognostic marker (Joyce, J. A.
  • CTSL inhibitors Although the therapeutic potential of CTSL inhibitors has not been fully characterized in preclinical studies, targeting CTSL activity is considered as a strategy for anticancer therapy (Lankelma, J.M. et al. 2010) . Therefore, drugs with improved selectivity are still urgently needed to combat cancer and various other diseases. Such selectivity allows for a drug with maximal efficacy and minimal adverse effects or toxicit .
  • the present invention provides a compound having the structure:
  • X is a therapeutic agent containing at least one amine nitogen and the amine nitrogen on the therapeutic agent covalently bonds directly to carbon a;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R4, R5, R6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 -e alkenyl, C 2 - 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • the present invention provides compound having the structure:
  • Y is a chemical linker containing at least one amine nitrogen
  • Y is a para-aminobenzyl alcohol linker
  • linker Y connects to the therapeutic agent X, or the oxygen on the linker Y connects directly to carbon a and the nitrogen on the linker Y connects to the therapeutic agent X through an amide bond;
  • Z is CH 3 or CF 3 ;
  • R 2 , R3, R4, R5, R6 and R 7 are each, independently,
  • Ci-6 alkyl C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • n is an integer from 0 to 6;
  • the invention provides a compound having the structure:
  • A is OH, 0(Ci-C 6 alkyl) or 0 (CH 2 -aryl) ;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 -e alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl, an amino acid or an oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • Figure 1 Comparative HDAC activity of the panel of cancer and normal cell lines measured by a standard HDAC assay using substrate Boc-Lys (Ac) -AMC either with vehicle control DMSO or TSA (1 ⁇ ) . Data represent mean values of triplicate measurements ⁇ s.d. RFU, relative fluorescent units.
  • Figure 2 Comparative live cell lysyl endopeptidase activity of the same cell lines as in Figure 1 using substrate Boc-Lys-AMC either with vehicle control DMSO or Z-FY-CHO (100 ⁇ ) . Data represent mean values of triplicate measurements ⁇ s.d. RFU, relative fluorescent units .
  • Figure 3 Prostate cancer cells exhibit high enzymatic activity to convert Boc-Lys (Ac) -AMC releasing AMC in live cells. Comparative live cell enzymatic activity.
  • Prostate cancer (PC-3, DU-145, and LNCaP) , colon cancer (HCT116 as a positive control sensitive to the drug) , and normal colon (CCD841-CoN as a negative control) cells were analyzed using substrate Boc-Lys (Ac) -AMC either with vehicle control DMSO, TSA (1 ⁇ ) or Z-FY-CHO ( ⁇ ) .
  • Data represent mean values of triplicate measurements ⁇ s.d. RFU, relative fluorescent units .
  • Data represents mean values of triplicate measurements ⁇ s.d. RFU, relative fluorescent units.
  • FIG. 5 A. HDAC assay using different substrates in BxPC3 cells (shGFP and shSki) . Cells were incubated with Boc-Lys (Ac) -AMC or Ac- Arg-Gly-Lys (Ac) -AMC (25 ⁇ ) for Jackpot with DMSO (vehicle) or TSA ( ⁇ ) as indicated, followed by cell lysis and trypsin treatment, and fluorescence measurements. Data represent mean values of triplicate measurements ⁇ standard deviation. RFU, relative fluorescent units.
  • FIG. 6 A. Release of AMC by combined HDAC and endogenous protease-dependent enzymatic reaction in BxPC3 and CFPac-1 cells (shGFP and shSki) . Cells were incubated with substrate Boc-Lys (Ac) - AMC (25 ⁇ ) for 6hr with DMSO (vehicle), TSA ( ⁇ ) , or Z-FY-CHO ( ⁇ ) as indicated, followed by fluorescence measurements. Data represent mean values of triplicate measurements ⁇ standard deviation. RFU, relative fluorescent units.
  • B Time course of AMC release in BxPC3 cells (shGFP and shSki) . Each cells were incubated with Boc-Lys (Ac) -AMC (25 ⁇ ) for 2.5, 6.0, and 23hr with DMSO (vehicle) or TSA ( ⁇ ) as indicated.
  • Figure 7. A scheme of the selective two-step drug activation in cancer cells by HDAC and CTSL.
  • Figure 8. Boc-Lys (Ac) -Puromycin, Spectral data for Boc-KAc-Puro.
  • FIG. 9 The colon cancer cell lines were grown for 5 d either with vehicle control DMSO (gray symbols) or 54 ⁇ BKAc-Puro (red and blue symbols), and cell number was determined at the indicated time points .
  • Figure 10 The cell lines were grown as in Figure 8, cell viability was determined by trypan blue at the indicated time points, and is presented as the percentage of live cells treated with vehicle control (gray symbols) or BKAc-Puro (red and blue symbols) .
  • Figure 11 The cell lines were grown as in Figure 8, and dead cells were determined after 80 h by PI signal under fluorescent microscope. BF, bright field. PI, fluorescent PI channel. Figure 12. Selective cytotoxicity by BKAc-Puro. The cell lines were grown and analyzed as in Figure 10. BF, bright field. PI , fluorescent PI channel .
  • Figure 13 Inhibition of cell viability by BKAc-Puro is presented in dose-response curve format for the same cell lines as in Figure 8.
  • the cell lines were treated with DMSO or the indicated doses of agent (4.22, 8.44, 16.9, 33.8, 67.5, or 135 ⁇ ) for 5 d followed by MTS assay.
  • IC 5 o values were fit by logistic regression.
  • Figure 14 Inhibition of cell viability by parental Puro.
  • the cell lines were treated with the indicated doses of Puro (0, 1.1, 2.1, 4.2, or 8.4 ⁇ ) for 3 d followed by MTS assay. Data represent mean values of triplicate measurements ⁇ s.d.
  • Figure 15 Inhibition of cell viability by BKAc-Puro.
  • the cell lines were grown and analyzed as in Figure 12.
  • FIG. 16 Anticancer effect of BKAc-Puro on pancreatic cell lines.
  • the cell lines were treated with DMSO (vehicle) , BKAc-Puro (54 ⁇ ) , or Puro (4.2 ⁇ ) for 5 d followed by MTS assay.
  • Normal Eph4 (nonpancreatic) cells are shown as control. Data represent mean values of triplicate measurements ⁇ s.d.
  • FIG. 17 Selective cytotoxicity by BKAc-Puro on breast cancer cells.
  • Normal mammary gland epithelial cells (Eph4) and breast cancer cells (MCF-7 and MDA-MB-231) were grown and analyzed as in Figure 10.
  • BF bright field.
  • PI fluorescent PI channel.
  • BKAc-Puro can selectively cause cell death in breast cancer cells (MCF-7 and MDA-MB-231) while leaving normal Eph4 cells unharmed.
  • FIG. 18 Selective cytotoxicity by BKAc-Puro on pancreatic cancer cells.
  • Pancreatic cancer BXPC-3 and Miapaca-2
  • colon cancer HCT116 as a positive control sensitive to the drug
  • normal colon CCD841-CoN as a negative control
  • BKAc-Puro can effectively cause cell death on pancreatic cancer cells (BXPC-3 and MiaPaca-2) that are known to resistant to conventional chemotherapeutic drugs including 5-FU and Gemicitabine .
  • FIG. 19 Selective cytotoxicity by BKAc-Puro on prostate cancer cells.
  • Prostate cancer PC-3, DU-145, and LNCaP
  • colon cancer HCT116 as a positive control sensitive to the drug
  • normal colon CCD841-CoN as a negative control
  • BKAc-Puro can effectively cause cell death on prostate cancer cells.
  • Figure 20 In vivo anticancer efficacy of BKAc-Puro.
  • DMSO vehicle control
  • FIG. 22 Dose escalation toxicity study.
  • Body weight change during the treatment is expressed in percent change compared to the day of the first treatment.
  • Arrows indicate the time points of treatment.
  • LD50 values for single administration of unmasked puromycin are 335 mg/kg (intravenously), 580 mg/kg (intraperitoneally) , and 720 mg/kg (orally) [ABANAE Antibiotics Annual, 1954/1955], BKAc-Puro appears to be well- protected and well-tolerated in animals.
  • FIG. 23 A. Synthesis of Boc-Lys (Ac ) -5-fluorocytidine (BKAc-5FCR) .
  • B Mechanism of prodrug activation by HDAC and tumor-associated protease .
  • FIG 24 Effect of the prodrug on cell viability of BxPC3 cells (shGFP and shSki) .
  • Cells were treated with the indicated dose of drugs (BKAc-5FCR or 5FCR) for 72hr followed by MTT assay.
  • Data represent mean values of triplicate measurements ⁇ standard deviation.
  • IC 50 values were derived from nonlinear curve fit of the dose response data using an outlier' s exclusion, variable slope model (GraphPad Prism software) . All values are means of at least two independent experiments.
  • Figure 25 Selective cytotoxicity by BKAc-5FCR.
  • Human pancreatic BXPC3 cells were treated with DMSO (vehicle), BKAc-5FCR (20 ⁇ ) , or 5FCR (20 ⁇ ) for 72hr with or without TSA (50nM) followed by MTS cell viability assay. Representative phase contrast images of cells treated with DMSO (vehicle) or BKAc-5FCR (20 ⁇ ) for 72hr with or without TSA (50nM) are shown. The HDAC-dependent activation of BKAc- 5FCR in BXPC-3 cells was further demonstrated in the presence of HDAC inhibitor TSA where the cytotoxic effect of BKAc-5FCR was substantially compromised.
  • FIG. 27 Levels of Puro-incorporated proteins in the cells were monitored by immunoblotting . Indicated cell lines were treated either with vehicle control DMSO or Boc-KAc-Puro (16.9 ⁇ ) in the presence or absence of TSA (0.5 ⁇ ) for 20 h, followed by preparation of cell lysates . The lower panel (anti- -Tubulin) serves as a loading control.
  • Figure 28 Levels of Puro-incorporated proteins in the cells were monitored by immunoblotting. Indicated cell lines were treated either with vehicle control DMSO or Puro (2.1 ⁇ ) in the presence or absence of TSA (0.5 ⁇ ) for 16 h, followed by preparation of cell lysates. The middle panel shows lighter exposure of the same blot in the top panel. The lower panel (anti-a-Tubulin) serves as a loading control .
  • FIG. 29 In vivo anticancer efficacy of Boc-KAc-Puro.
  • the present invention provides a compound having the structure:
  • X is a therapeutic agent containing at least one amine nitogen and the amine nitrogen on the therapeutic agent covalently bonds directly to carbon a;
  • Z is CH 3 or CF 3 ;
  • R 2 , R3, R4, R5, R6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 _ 6 alkenyl, C 2 -6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • the compound having the structure is:
  • X is a therapeutic agent containing at least one amine nitogen and the amine nitrogen on the therapeutic agent covalently bonds directly to carbon a;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R4, R5, R6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • the compound having the structure is:
  • X is a therapeutic agent containing at least one amine nitogen and the amine nitrogen on the therapeutic agent covalently bonds directly to carbon a;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 -6 alkenyl, C 2 -6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, an amino acid or an oligopeptide; wherein an amine of the amino acid or oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • R 2 , R 3 , R4, R5, R6 and R7 are each, independently,
  • Ci-6 alkyl C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is 4.
  • the compound wherein n is 3, 4 or 5. In some embodiments, the compound wherein
  • Rx is -NR 2 R 3 ,
  • R 2 is -H
  • R 3 is an amino acid
  • Ri is -NR 2 R 3 ,
  • R 2 is -H
  • R 3 is an oligopeptide
  • R 8 and Rg are each independently
  • the compound wherein X is a chemotherapeutic agent containing at least one amine nitrogen.
  • the compound wherein X is a nucleoside or deoxynucleoside containing at least one amine nitrogen.
  • the compound wherein X is puromycin, 5- fluorocytidine , 2 ' -deoxy-5-fluorocytidine, 5'-deoxy-5- fluorocytidine , 5' -deoxy-5-fluorocytidine, gemcitabine , cytarabine, cladribine, troxacitabine, adriamycin, alimta, aminolevulinic acid, azacitidine, bleomycin, cerubidine, clofarabine, clofarex, crizotinib, dasatinib, daunorubicin , decitabine, doxil, deoxorubicin , ellence, epirubicin, eribulin mesylate, erlotinib, evacet, fludara, fludarabine
  • the present invention provides compound having the structure:
  • X is a therapeutic agent
  • Y is a chemical linker
  • Y is a chemical linker containing at least one amine nitrogen
  • Y is a para-aminobenzyl alcohol linker
  • linker Y connects to the therapeutic agent X, or the oxygen on the linker Y connects directly to carbon a and the nitrogen on the linker ⁇ connects to the therapeutic agent X through an amide bond;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently,
  • Ci_ 6 alkyl C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • n is an integer from 0 to 6;
  • X is a therapeutic agent
  • Y is a chemical linker
  • Y is a chemical linker containing at least one amine nitrogen
  • Y is a para-aminobenzyl alcohol linker
  • linker Y connects to the therapeutic agent X, or the oxygen on the linker Y connects directly to carbon a and the nitrogen on the linker Y connects to the therapeutic agent X through an
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • n is an integer from 0 to 6;
  • X is a therapeutic agent
  • Y is a chemical linker
  • Y is a chemical linker containing at least one nitrogen
  • Y is a para-aminobenzyl alcohol linker
  • linker Y connects to the therapeutic agent X, or the oxygen on the linker Y connects directly to carbon a and the nitrogen on the linker Y
  • Z is CH 3 or CF 3 ;
  • R 2 , R3, R4, R5, R6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • n is an integer from 0 to 6;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently, -H, Ci_6 alkyl, C 2 -6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, alkylaryl, heteroaryl, alkylheteroaryl, an amino acid or an oligopeptide;
  • amine of the amino acid or oligopeptide is substituted or unsubstituted; and n is 4.
  • Y is present, and Y is a chemical linker containing at least one amine nitrogen,
  • Y is present, and Y is a para-aminobenzyl alcohol linker
  • the nitrogen on the linker Y connects to directly to carbon a and the oxygen on the linker Y connects to the therapeutic agent X, or the oxygen on the linker Y connects directly to carbon a and the nitrogen on the linker Y connects to the therapeutic agent X through an amide bond.
  • the compound wherein the oxygen on the linker Y connects directly to carbon a and the nitrogen on the linker Y connects to the therapeutic agent X through an amide bond.
  • the compound wherein n is 3, 4 or 5. In some embodiments, the compound wherein
  • Y is absent.
  • a nitrogen on the linker Y connects to directly to carbon a.
  • Ri is -NR 2 R 3 ,
  • R 2 is -H
  • R 3 is an amino acid
  • Ri is -NR 2 R 3 ,
  • R 2 is -H
  • R 3 is an oligopeptide
  • R 8 and R 9 are each independently
  • R 10 is -H, -CH 3 , Ac, -C (0) -Ot-Bu, -C(O)- OCH 2 Ph, -CHO, phenyl, or benzyl.
  • the compound wherein X is a chemotherapeutic agent .
  • the compound wherein X is a nucleoside or deoxynucleoside .
  • the compound wherein X is puromycin, 5- fluorocytidine , 2 ' -deoxy-5-fluorocytidine, 5'-deoxy-5- fluorocytidine , 5' -deoxy-5-fluorocytidine, gemcitabine, cytarabine, cladribine, troxacitabine , abiraterone acetate, abraxane, adriamycin, Armitor, alimta, aloxi, amboclorin, aminolevulinic acid, anastrozole, aprepitant, aromasin, axitinib, azacitidine, bendamustine hydrochloride, bexarotene, bleomycin, bortezomib, cabazitaxel, capecitabine, cerubidine, clofarabine, clofarex, crizotinib, dacarbazine, dasatinib, daunorubic
  • the compound wherein Z is CF 3 some embodiments, the compoun'
  • the compound having the structure having the structure
  • n 4 ;
  • X is puromycin or 5-fluorocytidine ; and Z is CH 3 .
  • a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier .
  • a method for reducing one or more symptoms of disease in a subject comprising administering an effective amount of the compound of the present invention or the composition of the present invention to the subject so as to treat the disease in the sub ect .
  • the disease is characterized by or caused by cells which have elevated levels of histone deacetylases or proteases or both.
  • the disease is cancer.
  • the compound or composition inhibits cancer cell metastasis.
  • the compound or composition inhibits cancer cell proliferation.
  • the cancer cells have elevated levels of histone deacetylases or proteases or both.
  • the cancer is colon, pancreatic, liver, breast, prostate, or cervical cancer.
  • a method for inhibiting growth of a tumor comprising contacting the tumor with a compound of the present invention or the composition of the present invention.
  • a method for reducing the size a tumor comprising contacting the tumor with a compound of the present invention or the composition of the present invention.
  • the compound or composition of the present invention for use in treating a subject suffering from cancer .
  • the compound or composition of the present invention for use in treating cancer.
  • the compound or composition of the present invention for use in inhibiting growth of a tumor.
  • the compound or composition of the present invention for use in reducing the size a tumor.
  • the compound or composition of the present invention for use in treating a disease that is caused by cells which have elevated levels of histone deacetylases or proteases or both .
  • the invention provides a compound having the structure:
  • A is OH, 0(Ci-C 6 alkyl) or 0 (CH 2 -aryl) ;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently, -H, Ci-6 alkyl, C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide; wherein an amine of the amino acid or oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • A is OH, 0(Ci-C 6 alkyl) or 0 (CH 2 -aryl) ;
  • Z is CH 3 or CF 3 ;
  • R 2 , R 3 , R 4 , R 5 , R 6 and R 7 are each, independently,
  • Ci-6 alkyl C 2 - 6 alkenyl, C 2 -6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • the compound having the structure having the structure
  • A is OH, 0(Ci-C 6 alkyl) or 0 (CH 2 -aryl) ;
  • Z is CH 3 or CF 3 ;
  • Ci_6 alkyl C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, aryl, or heteroaryl ,
  • R : R 3 , R 4 , R 5 , R 6 and R 7 are each, independently,
  • Ci_6 alkyl C 2 _ 6 alkenyl, C 2 _ 6 alkynyl, heteroalkyl, cycloalkyl, heterocyclyl , aryl, alkylaryl, heteroaryl, alkylheteroaryl , an amino acid or an oligopeptide;
  • oligopeptide is substituted or unsubstituted; and n is an integer from 0 to 6;
  • the invention provides a method of reducing one or more symptoms of any disease that involves carcinomas or cancer including but not limited to lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer; stomach cancer, esophagus cancer, mouth cancer, tongue cancer, gum cancer, skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi's sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testis cancer, kidney cancer, endometrium cancer, uterus cancer, bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymus cancer, thyroid cancer, brain cancer, neuron cancer, mesothelioma, gall bladder cancer, ocular cancer (e.g., cancer of the cornea, cancer of uvea, cancer of the choroids, cancer of the macula, vitreous humor cancer, etc.), joint cancer (such as synovium cancer),
  • amino acid refers to any natural or unnatural amino acid including its salt form, ester derivative, protected amine derivative and/or its isomeric forms.
  • Amino Acids comprise, by way of non-limiting example: Agmatine, Alanine Beta- Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Phenyl Beta-Alanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, and Valine.
  • the amino acids may be L or D amino acids.
  • oligopeptide refers to a peptide comprising of between 2 and 20 amino acids and includes dipeptides, tripeptides, tetrapeptides , pentapeptides , etc.
  • amino acid or oligopeptide may be covalently bonded to an amine of another molecule through an amide linkage, resulting in the loss of an "OH” from the amino acid or oligopeptide.
  • ara-aminobenzyl alcohol linker refers to
  • therapeutic agent refers to any agent used to treat a disease or that provides a beneficial therapeutic effect to a subject.
  • chemotherapeutic agent refers to any agent used to treat cancer or that provides a beneficial therapeutic effect to a subject suffering from cancer.
  • compositions or compounds containing therapeutic agents such as a cytotoxin, e.g., a cytostatic or cytocidal agent.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, coichicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone , mithramycin, actinomycin D, I-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil decarbazine) , alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU) , cyclothosphamide , busulfan, dibromomannitol , streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II), (DDP) cisplatin) , anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (fonnerly actinomycin) , bleomycin, mithramycin, and anthramycin) ,
  • compositions or compounds containing chemotherapeutic agents which are any agents detrimental to cancer cells.
  • chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mito-xantrone , amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercap
  • chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide) , or in combination with one or more other such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide) .
  • Anti-inflammatory drugs including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids
  • antiviral drugs including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir
  • ribivirin, vidarabine, acyclovir and ganciclovir may also be combined in compositions of the invention. See, generally, The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively.
  • Other chemotherapeutic agents are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.
  • the therapeutic or chemotherapeutic agent is not to be construed as limited to classical chemical therapeutic or chemotherapeutic agents.
  • the agent may be a protein, nucleotide or polypeptide possessing a desired biological activity.
  • therapeutic agent does not include fluorogenic probes, optical probes, radiolabeled probes, dyes, or other agents that function as imaging or contrast agents.
  • a histone deacetylase may be zinc- dependent.
  • HDACs include, but are not limited to, HDAC1, HDAC2 , HDAC3 , HDAC4, HDAC5, HDAC6, HDAC7 , HDAC8 , HDAC 9 , HDAC10, and HDAC11.
  • histone decetylases include, but are not limited to, the Sir2 proteins SIRTl, SIRT2, SIRT3, SIRT4, SIRT5, SIRT6 and SIRT7.
  • proteolysis refers to the hydrolysis or cleavage of a peptide or amide bond.
  • the term "activity" refers to the activation, production, expression, synthesis, intercellular effect, and/or pathological or aberrant effect of the referenced molecule, either inside and/or outside of a cell.
  • Such molecules include, but are not limited to, cytokines, enzymes, growth factors, pro-growth factors, active growth factors, and pro-enzymes. Molecules such as cytokines, enzymes, growth factors, pro-growth factors, active growth factors, and pro-enzymes may be produced, expressed, or synthesized within a cell where they may exert an effect. Such molecules may also be transported outside of the cell to the extracellular matrix where they may induce an effect on the extracellular matrix or on a neighboring cell.
  • inactive cytokines activation of inactive cytokines, enzymes and pro-enzymes may occur inside and/or outside of a cell and that both inactive and active forms may be present at any point inside and/or outside of a cell. It is also understood that cells may possess basal levels of such molecules for normal function and that abnormally high or low levels of such active molecules may lead to pathological or aberrant effects that may be corrected by pharmacological intervention.
  • This invention also provides isotopic variants of the compounds disclosed herein, including wherein the isotopic atom is 2 H and/or wherein the isotopic atom 13 C. Accordingly, in the compounds provided herein hydrogen can be enriched in the deuterium isotope. It is to be understood that the invention encompasses all such isotopic forms . It is understood that the structures described in the embodiments of the methods hereinabove can be the same as the structures of the compounds described hereinabove. It is understood that where a numerical range is recited herein, the present invention contemplates each integer between, and including, the upper and lower limits, unless otherwise stated.
  • each stereogenic carbon may be of the R or S configuration.
  • isomers arising from such asymmetry e.g., all enantiomers and diastereomers
  • Such isomers can be obtained in substantially pure form by classical separation techniques and by stereochemically controlled synthesis, such as those described in "Enantiomers, Racemates and Resolutions" by J. Jacques, A. Collet and S. Wilen, Pub. John Wiley & Sons, NY, 1981.
  • the resolution may be carried out by preparative chromatography on a chiral column.
  • the subject invention is also intended to include all isotopes of atoms occurring on the compounds disclosed herein.
  • Isotopes include those atoms having the same atomic number but different mass numbers.
  • isotopes of hydrogen include tritium and deuterium.
  • isotopes of carbon include C-13 and C-14.
  • any notation of a carbon in structures throughout this application when used without further notation, are intended to represent all isotopes of carbon, such as 12 C, 13 C, or C.
  • any compounds containing C or C may specifically have the structure of any of the compounds disclosed herein.
  • any notation of a hydrogen in structures throughout this application when used without further notation, are intended to represent all isotopes of hydrogen, such as X H, 2 H, or 3 H.
  • any compounds containing 2 H or 3 H may specifically have the structure of any of the compounds disclosed herein.
  • Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art using appropriate isotopically-labeled reagents in place of the non- labeled reagents employed.
  • the substituents may be substituted or unsubstituted, unless specifically defined otherwise.
  • alkyl, heteroalkyl, monocycle, bicycle, aryl, heteroaryl and heterocycle groups can be further substituted by replacing one or more hydrogen atoms with alternative non-hydrogen groups.
  • non-hydrogen groups include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl .
  • substituents and substitution patterns on the compounds used in the method of the present invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results . In choosing the compounds used in the method of the present invention, one of ordinary skill in the art will recognize that the various substituents , i.e. R lr R 2 , etc. are to be chosen in conformity with well-known principles of chemical structure connectivity.
  • alkyl includes both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and may be unsubstituted or substituted.
  • Ci-C n as in “Ci-C n alkyl” is defined to include groups having 1, 2, n-1 or n carbons in a linear or branched arrangement.
  • Ci-C 6 as in "Ci-C 6 alkyl” is defined to include groups having 1, 2, 3, 4, 5, or 6 carbons in a linear or branched arrangement, and specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, pentyl, hexyl, and octyl .
  • alkenyl refers to a non-aromatic hydrocarbon radical, straight or branched, containing at least 1 carbon to carbon double bond, and up to the maximum possible number of non- aromatic carbon-carbon double bonds may be present, and may be unsubstituted or substituted.
  • C 2 -C 6 alkenyl means an alkenyl radical having 2, 3, 4, 5, or 6 carbon atoms, and up to 1, 2, 3, 4, or 5 carbon-carbon double bonds respectively.
  • Alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl .
  • alkynyl refers to a hydrocarbon radical straight or branched, containing at least 1 carbon to carbon triple bond, and up to the maximum possible number of non-aromatic carbon-carbon triple bonds may be present, and may be unsubstituted or substituted.
  • C 2 -C 6 alkynyl means an alkynyl radical having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds, or having 6 carbon atoms and up to 3 carbon-carbon triple bonds.
  • Alkynyl groups include ethynyl, propynyl and butynyl .
  • Alkylene alkenylene and alkynylene shall mean, respectively, a divalent alkane, alkene and alkyne radical, respectively. It is understood that an alkylene, alkenylene, and alkynylene may be straight or branched. An alkylene, alkenylene, and alkynylene may be unsubstituted or substituted.
  • aryl is intended to mean any stable monocyclic, bicyclic or polycyclic carbon ring of up to 10 atoms in each ring, wherein at least one ring is aromatic, and may be unsubstituted or substituted.
  • aryl elements include phenyl, p- toluenyl (4-methylphenyl) , naphthyl, tetrahydro-naphthyl , indanyl, biphenyl, phenanthryl, anthryl or acenaphthyl .
  • the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that attachment is via the aromatic ring.
  • polycyclic refers to unsaturated or partially unsaturated multiple fused ring structures, which may be unsubstituted or substituted.
  • alkylaryl refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an aryl group as described above. It is understood that an “alkylaryl” group is connected to a core molecule through a bond from the alkyl group and that the aryl group acts as a substituent on the alkyl group.
  • arylalkyl moieties include, but are not limited to, benzyl (phenylmethyl ) , p-tri fluoromethylbenzyl (4- trifluoromethylphenylmethyl ) , 1-phenylethyl , 2-phenylethyl , 3- phenylpropyl , 2-phenylpropyl and the like.
  • heteroaryl represents a stable monocyclic, bicyclic or polycyclic ring of up to 10 atoms in each ring, wherein at least one ring is aromatic and contains from 1 to 4 heteroatoms selected from the group consisting of 0, N and S.
  • Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5- membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from 0, N or S.
  • Heteroaryl groups within the scope of this definition include but are not limited to: benzoimidazolyl , benzofuranyl, benzofurazanyl , benzopyrazolyl , benzotriazolyl , benzothiophenyl , benzoxazolyl , carbazolyl, carbolinyl, cinnolinyl, furanyl, indolinyl, indolyl, indolazinyl, indazolyl, isobenzofuranyl , isoindolyl, isoquinolyl, i sothiazolyl , isoxazolyl, naphthpyridinyl , oxadiazolyl, oxazolyl, oxazoline, isoxazoline, oxetanyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridopyridinyl , pyridaziny
  • heteroaryl substituent is bicyclic and one ring is non-aromatic or contains no heteroatoms, it is understood that attachment is via the aromatic ring or via the heteroatom containing ring, respectively. If the heteroaryl contains nitrogen atoms, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
  • alkylheteroaryl refers to alkyl groups as described above wherein one or more bonds to hydrogen contained therein are replaced by a bond to an heteroaryl group as described above. It is understood that an "alkylheteroaryl” group is connected to a core molecule through a bond from the alkyl group and that the heteroaryl group acts as a substituent on the alkyl group. Examples of alkylheteroaryl moieties include, but are not limited to, -CH 2 - (C 5 H 4 N) , -CH 2 -CH 2 - (C 5 H 4 N) and the like.
  • heterocycle refers to a mono- or poly-cyclic ring system which can be saturated or contains one or more degrees of unsaturation and contains one or more heteroatoms.
  • Preferred heteroatoms include N, 0, and/or S, including N-oxides, sulfur oxides, and dioxides.
  • the ring is three to ten-membered and is either saturated or has one or more degrees of unsaturation.
  • the heterocycle may be unsubstituted or substituted, with multiple degrees of substitution being allowed.
  • Such rings may be optionally fused to one or more of another "heterocyclic" ring(s), heteroaryl ring(s), aryl ring(s), or cycloalkyl ring(s) .
  • heterocycles include, but are not limited to, tetrahydrofuran , pyran, 1,4-dioxane, 1,3-dioxane, piperidine, piperazine, pyrrolidine, morpholine, thiomorpholine, tetrahydrothiopyran, tetrahydrothiophene, 1 , 3-oxathiolane, and the like.
  • alkyl, alkenyl, alkynyl, aryl, heteroaryl and heterocyclyl substituents may be substituted or unsubstituted, unless specifically defined otherwise.
  • alkyl, alkenyl, alkynyl, aryl, heterocyclyl and heteroaryl groups can be further substituted by replacing one or more hydrogen atoms with alternative non- hydrogen groups.
  • hydrogen atoms include, but are not limited to, halo, hydroxy, mercapto, amino, carboxy, cyano and carbamoyl.
  • halogen refers to F, CI, Br, and I.
  • heteroalkyl includes both branched and straight- chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms and at least 1 heteroatom within the chain or branch .
  • heterocycle or “heterocyclyl” as used herein is intended to mean a 5- to 10-membered nonaromatic ring containing from 1 to 4 heteroatoms selected from the group consisting of 0, N and S, and includes bicyclic groups.
  • Heterocyclyl therefore includes, but is not limited to the following: imidazolyl, piperazinyl, piperidinyl, pyrrolidinyl , morpholinyl, thiomorpholinyl , tetrahydropyranyl , dihydropiperidinyl , tetrahydrothiophenyl and the like. If the heterocycle contains a nitrogen, it is understood that the corresponding N-oxides thereof are also encompassed by this definition.
  • cycloalkyl shall mean cyclic rings of alkanes of three to eight total carbon atoms, or any number within this range (i.e., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl) .
  • monocycle includes any stable polyatomic carbon ring of up to 10 atoms and may be unsubstituted or substituted.
  • non-aromatic monocycle elements include but are not limited to: cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • aromatic monocycle elements include but are not limited to: phenyl.
  • bicycle includes any stable polyatomic carbon ring of up to 10 atoms that is fused to a polyatomic carbon ring of up to 10 atoms with each ring being independently unsubstituted or substituted.
  • non-aromatic bicycle elements include but are not limited to: decahydronaphthalene .
  • aromatic bicycle elements include but are not limited to: naphthalene .
  • esters is intended to a mean an organic compound containing the R-O-CO-R' group.
  • amide is intended to a mean an organic compound containing the R-CO-NH-R' or R-CO-N-R'R” group.
  • phenyl is intended to mean an aromatic six membered ring containing six carbons and five hydrogens.
  • benzyl is intended to mean a -CH 2 Ri group wherein the Ri is a phenyl group.
  • substitution refers to a functional group as described above in which one or more bonds to a hydrogen atom contained therein are replaced by a bond to non- hydrogen or non-carbon atoms, provided that normal valencies are maintained and that the substitution results in a stable compound.
  • Substituted groups also include groups in which one or more bonds to a carbon (s) or hydrogen (s) atom are replaced by one or more bonds, including double or triple bonds, to a heteroatom.
  • substituent groups include the functional groups described above, and halogens (i.e., F, CI, Br, and I); alkyl groups, such as methyl, ethyl, n-propyl, isopropryl, n-butyl, tert-butyl, and trifluoromethyl ; hydroxyl; alkoxy groups, such as methoxy, ethoxy, n-propoxy, and isopropoxy; aryloxy groups, such as phenoxy; arylalkyloxy, such as benzyloxy (phenylmethoxy) and p- trifluoromethylbenzyloxy (4-trifluoromethylphenylmethoxy) ; heteroaryloxy groups; sulfonyl groups, such as trifluoromethanesulfonyl , methanesulfonyl, and p-toluenesulfonyl; nitro, nitrosyl; mercapto; sulf
  • substituted compound can be independently substituted by one or more of the disclosed or claimed substituent moieties, singly or plurally.
  • independently substituted it is meant that the (two or more) substituents can be the same or different.
  • nucleosides refers to glycosylamines consisting of a nucleobase bound to a ribose or deoxyribose sugar via a beta- glycosidic linkage. Such “nucleosides” may be naturally occurring or synthetic. Examples of such nucleosides are, but not limited to, 5- fluorocytidine and cytarabine. As used herein, “dexoynucleosides” refers to nucleosides with at least one less oxygen atom. Such “deoxynucleosides” may be naturally occurring or synthetic. Examples of such deoxynucleosides are, but not limited to, 5 ' -deoxy-5-fluorouridine and 2'-deoxy-5- fluorocytidine .
  • substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art, as well as those methods set forth below, from readily available starting materials. If a substituent is itself substituted with more than one group, it is understood that these multiple groups may be on the same carbon or on different carbons, so long as a stable structure results.
  • the compounds used in the method of the present invention may be prepared by techniques well known in organic synthesis and familiar to a practitioner ordinarily skilled in the art. However, these may not be the only means by which to synthesize or obtain the desired compounds.
  • the compounds used in the method of the present invention may be prepared by techniques described in Vogel's Textbook of Practical
  • Another aspect of the invention comprises a compound used in the method of the present invention as a pharmaceutical composition.
  • a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier .
  • the term "pharmaceutically active agent” means any substance or compound suitable for administration to a subject and furnishes biological activity or other direct effect in the treatment, cure, mitigation, diagnosis, or prevention of disease, or affects the structure or any function of the subject.
  • Pharmaceutically active agents include, but are not limited to, substances and compounds described in the Physicians' Desk Reference (PDR Network, LLC; 64th edition; November 15, 2009) and "Approved Drug Products with Therapeutic Equivalence Evaluations" (U.S. Department Of Health And Human Services, 30 th edition, 2010), which are hereby incorporated by reference.
  • compositions which have pendant carboxylic acid groups may be modified in accordance with the present invention using standard esterification reactions and methods readily available and known to those having ordinary skill in the art of chemical synthesis. Where a pharmaceutically active agent does not possess a carboxylic acid group, the ordinarily skilled artisan will be able to design and incorporate a carboxylic acid group into the pharmaceutically active agent where esterification may subsequently be carried out so long as the modification does not interfere with the pharmaceutically active agent's biological activity or effect.
  • the compounds used in the method of the present invention may be in a salt form.
  • a “salt” is a salt of the instant compounds which has been modified by making acid or base salts of the compounds.
  • the salt is pharmaceutically acceptable.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as phenols.
  • the salts can be made using an organic or inorganic acid.
  • Such acid salts are chlorides, bromides, sulfates, nitrates, phosphates, sulfonates, formates, tartrates, maleates, malates, citrates, benzoates, salicylates, ascorbates, and the like.
  • Phenolate salts are the alkaline earth metal salts, sodium, potassium or lithium.
  • pharmaceutically acceptable salt in this respect, refers to the relatively non-toxic, inorganic and organic acid or base addition salts of compounds of the present invention.
  • salts can be prepared in situ during the final isolation and purification of the compounds of the invention, or by separately reacting a purified compound of the invention in its free base or free acid form with a suitable organic or inorganic acid or base, and isolating the salt thus formed.
  • Representative salts include the hydrobromide , hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate , and laurylsulphonate salts and the like. (See, e.g., Berge et al . (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
  • treating means preventing, slowing, halting, or reversing the progression of a disease or infection. Treating may also mean improving one or more symptoms of a disease or infection.
  • the compounds used in the method of the present invention may be administered in various forms, including those detailed herein.
  • the treatment with the compound may be a component of a combination therapy or an adjunct therapy, i.e. the subject or patient in need of the drug is treated or given another drug for the disease in conjunction with one or more of the instant compounds.
  • This combination therapy can be seguential therapy where the patient is treated first with one drug and then the other or the two drugs are given simultaneously. These can be administered independently by the same route or by two or more different routes of administration depending on the dosage forms employed.
  • a "pharmaceutically acceptable carrier” is a pharmaceutically acceptable solvent, suspending agent or vehicle, for delivering the instant compounds to the animal or human.
  • the carrier may be liguid or solid and is selected with the planned manner of administration in mind.
  • Liposomes are also a pharmaceutically acceptable carrier.
  • the dosage of the compounds administered in treatment will vary depending upon factors such as the pharmacodynamic characteristics of a specific chemotherapeutic agent and its mode and route of administration; the age, sex, metabolic rate, absorptive efficiency, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment being administered; the frequency of treatment with; and the desired therapeutic effect.
  • a dosage unit of the compounds used in the method of the present invention may comprise a single compound or mixtures thereof with additional antibacterial agents.
  • the compounds can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • the compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by injection, topical application, or other methods, into or onto a site of infection, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts .
  • the compounds used in the method of the present invention can be administered in admixture with suitable pharmaceutical diluents, extenders, excipients, or carriers (collectively referred to herein as a pharmaceutically acceptable carrier) suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • a pharmaceutically acceptable carrier suitably selected with respect to the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the unit will be in a form suitable for oral, rectal, topical, intravenous or direct injection or parenteral administration.
  • the compounds can be administered alone or mixed with a pharmaceutically acceptable carrier.
  • This carrier can be a solid or liquid, and the type of carrier is generally chosen based on the type of administration being used.
  • the active agent can be co-administered in the form of a tablet or capsule, liposome, as an agglomerated powder or in a liquid form.
  • suitable solid carriers include lactose, sucrose, gelatin and agar.
  • Capsule or tablets can be easily formulated and can be made easy to swallow or chew; other solid forms include granules, and bulk powders. Tablets may contain suitable binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • liquid dosage forms examples include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifyinq agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Oral dosage forms optionally contain flavorants and coloring agents.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • Tablets may contain suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents, and melting agents.
  • the active drug component can be combined with an oral, non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methyl cellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.
  • Suitable binders include starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.
  • the compounds used in the method of the present invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamallar vesicles, and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.
  • the compounds may be administered as components of tissue-targeted emulsions.
  • the compounds used in the method of the present invention may also be coupled to soluble polymers as targetable drug carriers or as a prodrug.
  • soluble polymers include polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol , polyhydroxyethylasparta-midephenol , or polyethyleneoxide-polylysine substituted with palmitoyl residues.
  • the compounds may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters , polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
  • a class of biodegradable polymers useful in achieving controlled release of a drug
  • a drug for example, polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters , polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels.
  • Gelatin capsules may contain the active ingredient compounds and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as immediate release products or as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • liquid dosage form For oral administration in liquid dosage form, the oral drug components are combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • water a suitable oil, saline, aqueous dextrose (glucose) , and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium EDTA are also used.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • preservatives such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol .
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field.
  • the compounds used in the method of the present invention may also be administered in intranasal form via use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will generally be continuous rather than intermittent throughout the dosage regimen.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • the compounds and compositions of the present invention can be administered in oral dosage forms as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions.
  • the compounds may also be administered in intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, or introduced directly, e.g. by topical administration, injection or other methods, to the afflicted area, such as a wound, including ulcers of the skin, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
  • prodrug refers to any compound that when administered to a biological system generates the compound of the invention, as a result of spontaneous chemical reaction (s), enzyme catalyzed chemical reaction(s), photolysis, and/or metabolic chemical reaction (s) .
  • a prodrug is thus a covalently modified analog or latent form of a compound of the invention.
  • the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, powders, and chewing gum; or in liquid dosage forms, such as elixirs, syrups, and suspensions, including, but not limited to, mouthwash and toothpaste. It can also be administered parentally, in sterile liquid dosage forms. Solid dosage forms, such as capsules and tablets, may be enteric coated to prevent release of the active ingredient compounds before they reach the small intestine.
  • Materials that may be used as enteric coatings include, but are not limited to, sugars, fatty acids, waxes, shellac, cellulose acetate phthalate (CAP) , methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate) , polyvinyl acetate phthalate (PVAP) , and methyl methacrylate- methacrylic acid copolymers.
  • CAP cellulose acetate phthalate
  • PVAP polyvinyl acetate phthalate
  • the compounds and compositions of the invention can be coated onto stents for temporary or permanent implantation into the cardiovascular system of a subject.
  • a-Boc-Lys ( ⁇ -Ac ) -OH and puromycin dihydrochloride were purchased from Bachem and TOKU-E, respectively, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC-HC1) and 1-hydroxybenzotriazole (HOBt-H20) were purchased from Advanced Chem Tech.
  • DCM dichloromethane
  • DMF dimethylformamide
  • DMF dimethylformamide
  • DMF was dried and purified by solvent pushstill (SG water USA LLC, Nashua, NH) .
  • 1 E NMR data are reported as chemical shift in ppm (multiplicity, coupling constant in Hz, integration, and tentative assignment.
  • 13 C NMR data are reported as chemical shift in ppm (tentative assignment) . Assignments are based on expected chemical shifts, multiplicities, and coupling constants.
  • Colon cancer (Caco-2, COGA-10, HCA-7, HT29, HCT116), normal colon CCD841-CoN, pancreatic cancer (MiaPaca-2, BXPC-3), liver cancer HepG2, and cervical cancer HeLa and normal mouse mammary epithelial Eph4 cell lines were maintained in Iscove's Modified Dulbecco ' s Medium (IMDM, Invitrogen) supplemented with 10% fetal bovine serum and 100 U/ml penicillin/ streptomycin at 37 °C with 5 % CO 2 atmosphere .
  • IMDM Iscove's Modified Dulbecco ' s Medium
  • HDAC substrate 25 ⁇ Boc-Lys (Ac) -AMC either with DMSO or 1 ⁇ TSA (Sigma)] was applied to the overnight culture seeded from 6 x 104 cells in 100 ⁇ medium in 96-well tissue culture plates, followed by 2 to 3 h incubation at 37 °C with 5 % CO 2 atmosphere.
  • Live cell lysyl endopeptidase assay was performed by applying the substrate 25 ⁇ Boc-Lys-AMC (Bachem) [either with DMSO or 100 ⁇ Z-FY-CHO (EMD) ] to the overnight culture seeded from 6 x 104 cells in 100 ⁇ medium in 96-well tissue culture plates, followed by 20 h incubation at 37 °C with 5 % CO2 atmosphere, then the fluorescent signal of AMC was measured.
  • Live cell enzymatic assay using Boc-Lys (Ac) -AMC was performed by applying the substrate 25 ⁇ Boc-Lys (Ac) -AMC (either with DMSO, ⁇ TSA or 100 ⁇ Z-FY-CHO) to the overnight culture seeded from 6 x 104 cells in 100 ⁇ medium in 96-well tissue culture plates, followed by 20 h incubation at 37 °C with 5 % C0 2 atmosphere, then the fluorescent signal of AMC was measured. Experiments were repeated at least three times.
  • Cell number was determined by cell counting using improved Neubauer hemacytometer.
  • Cell viability was calculated as the number of viable cells divided by the total number of cells using Trypan Blue Stain (Invitrogen) to distinguish non-viable cells. Data were obtained from duplicated samples with quadruplicate measurements.
  • MTS based cell viability assay was performed using CellTiter 96 Aqueous Cell Proliferation Assay (Promega) according to the manufacturer's instruction.
  • Either 1 ⁇ of DMSO alone or variable concentrations of BKAc-Puro or Puro in DMSO were added to the cell lines seeded at 5 x 104 cells per well in 96-well tissue culture plates in 100 ⁇ of the growth medium, followed by 3-5 d incubation at 37 °C with 5 % C02 atmosphere. Then 20 ⁇ of the MTS reagent was added to each well. After additional 2-3 h incubation, the absorbance of the formazan at 490 nm was measured by the microplate reader. Percent cell viability was expressed relative to the wells containing cells treated with DMSO alone. Data were obtained from triplicate measurements.
  • IC 50 values the concentration resulting in 50% inhibition of BKAc-Puro were determined by dose-response curve analysis (GraphPad Prism software) . Experiments were repeated at least three times. Determination of non-viable cells using PI staining was performed by adding 1 g/ml of PI solution (Sigma) to the cell culture, prior to the examination under fluorescence microscopy (Axiovert 3, Carl Zeiss) through a x 32 objective equipped with a digital imaging processor (Infinity 3, Lumenera) .
  • a BxPC3 xenograft model is used as an established evaluation method for the anticancer drugs against pancreatic cancer (Kano, M.R. et al. 2007 ; O'Toole, J.M. et al . 200 6 ) .
  • the cells are transfected with expression plasmids for GFP and selected for GFP expression in order to mark the tumor cells.
  • the growth inhibitory effects of the compounds on size-matched BxPC3 xenografts are examined by subcutaneous implantation of BxPC3 cells into nude mice. BxPC3 cells are injected subcutaneously and allowed to grow for 2-3 weeks to reach proliferative phase before initiation of drug administration.
  • mice (5 animals per group) are treated with the vehicle (PBS) , the various prodrugs, or 5-FU by intraperitoneal administration every 3 days for a total of 4 doses.
  • the prodrugs are expected to have lower toxicity than the parental 5-FU, thus dose ranges that have been established by others for 5-FU are used (Overholser, J. P. et al . 2000) .
  • the mice are imaged over a 3 week period using the Maestro small animal imaging system. This scanner captures tumor growth in the mice by detection of increasing area and intensity of green fluorescence emitted from the GFP.
  • Relative tumor volume is calculated by dividing tumor volume by that on day 0 (the day of treatment initiation) .
  • the weight of the mice is checked to monitor unfavorable effects by the compounds being tested and upon any indication of distress the mice is humanely euthanized.
  • Statistical significance of the data is evaluated by performing oneway ANOVA with post hoc Turkey' s test to compare means (GraphPad Prism software) .
  • the efficacy of the compounds is further evaluated in a similar manner by using other pancreatic adenocarcinoma cell lines CFPac-1 and MiaPaCa-2 and the PDA lines in which HDAC3 levels have been reduced.
  • mouse xenograft model using HCT116 colon cancer cells is used as an established evaluation method for the anticancer drugs against cancer (Cao, Z.A. et al . 2006) .
  • the growth inhibitory effects of the compounds on size-matched xenografts are examined by subcutaneous implantation of HCT116 cells into nude mice.
  • HCT116 cells are injected subcutaneously and allowed to develop palpable tumors (50-150 mm 3 ) before initiation of drug administration.
  • Mice (5-10 animals per group) are treated with the vehicle (DMSO or PBS) or the prodrugs by intraperitoneal administration. Dose ranges are determined by the dose escalation studies of the prodrugs.
  • Relative tumor volume is calculated by dividing tumor volume by that on day 0 (the day of treatment initiation) .
  • the weight of the mice is checked to monitor unfavorable effects by the compounds being tested and upon any indication of distress the mice is humanely euthanized.
  • Statistical significance of the data is evaluated by performing one-way ANOVA with post hoc Turkey's test to compare means (GraphPad Prism software) .
  • the efficacy of the compounds is further evaluated in a similar manner by using other cancer cell lines.
  • Athymic mice (NCr, female, age 6 weeks, Taconic) were subcutaneously injected with 5 x 10 5 cells (HCT116 or HT29) into the lower flank, then treatment was initiated when small palpable tumors had developed (> 3 mm in diameter) .
  • HDAC activity was measured in a panel of human cancer cell lines including colon cancer (Caco-2, COGA-10, HCA-7, HT29, HCT116), pancreatic cancer (BXPC-3, MiaPaca-2), liver cancer (HepG2), and cervical cancer (HeLa) , as well as non-tumorigenic human colon epithelial cells (CCD841-CoN) and normal mouse mammary epithelial cells (Eph4) (Mariadason, J.M. et al. 2001; Wegener, D. et al. 2003) ( Figure 1) .
  • HDAC HDAC inhibitor Trichostatin A
  • Caco-2 cells originated from human colon adenocarcinoma, these cells are known to be less-tumorigenic and behave like normal, differentiated enterocytes in vivo when cultured as confluent cells (Mariadason, J.M. et al . 2001) .
  • Typical fluorogenic substrates for class I HDACs are comprised of an ⁇ -acetylated lysine residue (alone or in short peptides) coupled to a fluorophor moiety AMC ( 7-amino-4-methylcoumarin) such as cell- permeable Boc-Lys (Ac) -AMC (Wegener, D. et al . 2003; Bonfils, C. et al . 2008) .
  • AMC 7-amino-4-methylcoumarin
  • the standard HDAC assay is based on the two-step conversion of the substrate: 1) HDAC-dependent deacetylation of ⁇ - acetylated lysine, 2) protease-dependent cleavage of Lys-AMC amide bond and subsequent release of free fluorophor AMC, which fluoresces (Wegener, D. et al . 2003).
  • the second step is normally performed following cell lysis by addition of excessive amounts of trypsin, which only recognizes and processes deacetylated form of lysine.
  • Lys-AMC amide bond can be cleaved by other endogenous proteases commonly associated with cancer cells such as lysosomal proteases (Weissleder, R. et al. 1999), it was hypothesized that the second step can occur in live cells without cell lysis and trypsin treatment.
  • CTSL lysosomal cysteine protease catepsin L
  • substrates for the sensitive fluorogenic assay of class I HDACs are comprised of a short peptide sequences coupled with an ⁇ -acetylated lysine residue followed by a fluorophor moiety (Wegener, D. et al 2003) .
  • substrates include Boc-Lys (Ac ) -X and Ac-Arg-Gly-Lys (Ac) -X, where X represents fluorophor moiety such as AMC.
  • the assay was also based on the two-step conversion of the fluorogenic peptide substrate: 1) Intracellular HDAC-dependent deacetylation of ⁇ -acetylated lysil moiety, 2) protease-dependent cleavage of Lys-X amide bond and subsequent release of free fluorophor.
  • the second step required cell lysis followed by addition of excessive amounts of exogenous proteases (commonly trypsin) to the reaction.
  • FIG. 4A shows the standard assay using these compounds, which were incubated with cells for various times, in this case 2.5 hr, during which time they were readily taken up by the cells and the endogenous HDAC enzymes deacetylate the lysine residues. Cells were then lysed and the lysates incubated with exogenously added excess trypsin to cleave only those lysine residues that are de- acetylated and activate fluorescence.
  • FIG. 5 shows a comparison of the normal assay (A) in which cells are lysed and then incubated with trypsin with a similar experiment (B) in which live cells were incubated with the same fluorescent compounds.
  • BKAc-Puro exhibits high grade of selectivity toward cells with high HDAC and CTSL activity, which is the characteristic of malignant cancer, while securing tight protection to non-malignant and normal cells with low (basal) HDAC and CTSL activity.
  • BKAc-Puro selectively caused cell death in breast cancer cells (MCF- 7 and MDA-MB-231) while leaving normal mammary gland epithelial cells (Eph4) unharmed (Figure 17).
  • BKAc-Puro effectively caused cell death in pancreatic cancer cells (BXPC-3 and MiaPaca-2) that are known to resistant to conventional chemotherapeutic drugs including 5-FU and Gemicitabine while having relatively little effect on normal colon cells unharmed (Figure 18) .
  • BKAc-Puro effectively caused cell death in prostate cancer cells (PC-3, DU-145 and LNCaP) while having relatively little effect on normal colon cells unharmed (Figure 19) .
  • Lys (Ac) -5-fluorocytdine) A mixture of 5-fluorocytidine, N- - (t- butoxycarbonyl ) - ⁇ - ⁇ -acetyl-L-lysine (Boc-Lys (Ac) -OH) , l-ethyl-3- (3- dimethylaminoproryl ) carbodiimide hydorochloride (EDC-HC1), 1- hydorxybenzotriazole (HOBt) , and N, N-diisopropylethylamine were stirred in DMF (dimethylformamide) for 18 h. Water was then added and the organic soluble part was extracted with methylene chloride. Then the compound was purified by silica gel column chromatography using 5-10 % MeOH in methylene chloride and dried to give Boc- Lys (Ac ) -5-fluorocytidine .
  • IC 50 values were determined for compound Boc-Lys (Ac ) -5-fluorocytidine (BKAc-5FCR) and it's parental drug 5FCR in BxPC3 cells stably expressing shRNas against either GFP or Ski (shGFP and shSki) . Consistent with the 50-60% difference in HDAC activity in these cells ( Figures 4-5), a moderate but significant reduction in drug response was seen in BxPC3 shSki cells treated with BKAc-5FCR over shGFP control cells, ( Figure 24), which showed a two-fold difference in IC 50 values, shSki: 5.2 versus shGFP: 2.6. In contrast, no significant changes in IC 50 values were observed in the same cells treated with 5FCR (shSki: 1.2 versus shGFP: 1.3) .
  • pancreatic cancer cell lines such as BxPC3 and CFPac-1 cells
  • kinetic parameters of cleavage of the following available substrates are analyzed for cathepsin L conjugated to AMC : Z-Phe- Lys-, Boc-Phe-Lys- , Ac-Phe-Lys-, HCO-Phe-Lys- , Z-Lys-, Boc-Lys-, Ac- Lys-, HCO-Lys-, where Z (benzyloxy-carbonyl), Boc (tert- butoxycarbonyl ) , Ac (acetyl), and HCO (formyl) represent -amino protecting groups .
  • An additional aspect of the invention provides compounds with variable peptide substrates and variable parental amine-containing and non-amine containing nucleosides and deoxynucleosides including 2 ' -deoxy-5-fluorocytidine, 5 ' -deoxy-5-fluorocytidine (precursors of 5-FU) , Gemicitabine , and Cytarabine ( Figure 26) .
  • Such compounds are synthesized by a similar amide coupling and are expected to function analogously to compound BKAc-5FCR and BKAc-Puro.
  • An additional aspect of the invention provides compounds with variable peptide substrates and variable parental amine-containing containing chemotherapeutic agents and are synthesized by a similar amide coupling. Such compounds are expected to function analogously to compound BKAc-5FCR and BKAc-Puro.
  • An additional aspect of the invention provides compounds with variable peptide substrates and variable non-amine containing chemotherapeutic agents and are synthesized by an amide coupling of the peptide substrate to a "Y" linker which is attached to the chemotherapeutic agent.
  • Compounds with a variety of "Y” linkers are expected to function analogously to compound BKAc-5FCR and BKAc- Puro.
  • the linker "Y” may be a "self-immolative” linker, which cleaves spontaneously after the carrier-linker bond, i.e. the amide bond, is broken.
  • Example of such a "self-immolative” linker includes, nut is not limited to, a para-aminobenzyl alcohol linker (Richard, J. et al . 2008) .
  • An additional aspect of the invention provides compounds with variable peptide substrates and variable chemotherapeutic agents that are customized to act as selective substrates for any of the specific HDACi, e.g., HDAC3.
  • An additional aspect of the invention provides compounds with variable peptide substrates and variable therapeutic agents that are customized to act as therapeutics for any disease that is associated with elevated levels of HDACs, proteases or both,
  • diseases include, but are not limited to, neurodegenerative diseases (Chuang, D. et al . 2009), Alzheimers disease, Parkinson's disease, neuropsychiatric diseases (Fischer, A. et al . 2010), infectious disease such as HIV/AIDS (Andrew, K.T. et al . 2012), parasitic diseases (Andrew, K.T. et al . 2012) or inflammatory diseases (Halili, M.A. et al. 2009) .
  • Example 7 Additional Biological Studies
  • the prodrugs are evaluated by determining IC 50 values in other cancer cell lines. It is expected that cells which have the lowest HDAC levels, either due to improved knockdown of Ski levels or as a direct result of shRNA directed against HDAC3 (and potentially other HDACs) would be more resistant to the prodrugs. Conversely, it is expected that cells which have the highest HDAC levels would be less resistant to the prodrugs. Monitoring of Prodrugs by HPLC
  • prodrugs are processed by HDACs and cathepsin L
  • commercially available purified enzymes class I HDACs and cathepsin L are available from TEBU-BIO
  • HPLC C-18 column
  • TLC TLC
  • hydrolysis studies are performed by measuring enzymatic and non-enzymatic stability of the prodrugs in human plasma, tissue culture medium containing 20% fetal calf serum, and defined buffer solutions with variable pH.
  • mice xenograft models bearing human colon cancer cell lines were used (HCT116 and HT29) .
  • HCT116 cells were subcutaneously injected into the lower flank of mice, then dosing was initiated when small palpable tumors had developed (> 3 mm in diameter) .
  • Boc-KAc-Puro was daily administered intraperitoneally at 50 and 150 mg/kg/dose for 10 d.
  • the agent caused a dose-dependent inhibition of tumor growth (Figure 29A) .
  • animals treated with the prodrug developed significantly smaller tumor mass in comparison to the animals treated with acidified saline control (P ⁇ 0.001) .
  • Histone deacetylases are the key enzymes involved in the epigenetic regulation of histone and non-histone proteins by modulating protein structure and function through deacetylation of lysine residues (Witt, 0. et al . 2009). Protein lysine acetylation is tightly regulated by HDACs and histone acetyltransferases (HATs), which influence chromatin dynamics, protein turnover and DNA damage response (Witt, 0. et al . 2009; Lee, K.K. & Workman, J.L. 2007; Choudary, C. et al. 2009) . Thus disregulation of these enzymes could lead to a broad spectrum of human diseases including cancer.
  • HDACs histone acetylases
  • HDAC inhibitors Minucci, S. et al . 2006
  • intrinsically elevated HDAC activity can be taken advantage of in order to selectively deliver cytotoxicity to the tumor cells. Since certain tumor cells are reliant on their elevated levels of HDACs to survive and proliferate under stressful conditions, a chemical HDAC substrate coupled to a therapeutic agent can preferentially cause lethality in cells with high, but not low, HDAC activity.
  • the HDAC substrate Upon deacetylation by HDAC, the HDAC substrate is, in turn, recognized as a substrate by specific intracellular proteases that cleave amide bonds, which ultimately results in release of the therapeutic agent.
  • HDAC activity is undetectable in plasma, and the amide bond between HDAC substrate and therapeutic agent is hardly cleaved by ubiquitous proteases in cytoplasm or plasma, this approach also minimizes the known drawbacks of peptide-based prodrugs .
  • HDAC histone deacetylase
  • the prodrug itself or the therapeutic agent X should not be an HDACi or protease inhibitor. This approach is promising strategy for the next generation of selective anticancer drugs.
  • nucleosides such as gemicitabine (Heunemann, V. et al . 2011) and 5-FU ( 5- fluorouracil ) (Lamont, E.B. et al. 1999) are current standard therapeutic regimens for pancreatic tumors, their efficacy is far from an ideal treatment for this devastating disease. Thus, there is an urgent need to develop drugs that are more effective than these conventional drugs. As explained herein, elevated HDAC activity, which is mediated by Ski oncoprotein in pancreatic ductal adenocarcinoma (PDA) can provide a therapeutic target . Selective prodrugs incorporating nucleoside parent agents were designed and synthesized.
  • prodrugs were synthesizied by coupling "nonselective" nucleoside antimetabolite analogs such as 5- fluorocytidine and puromycin to HDAC substrates to produce prodrugs BKAc-5FCR and BKAc-puro, respectively.
  • the prodrugs were activated by HDACs and proteases, which resulted in selective delivery of the parent drug to the cancer cells.
  • the prodrugs that are synthesized may have some basal level of cytotoxicity before they are activated by HDAC3.
  • One way to determine the IC 50 values of the uncleaved forms of the prodrugs would be to use HDAC inhibitors both in vitro and in vivo.
  • TSA was used to inhibit HDACs in the cells, unfortunately due to the cytotoxic effect of TSA at ⁇ over the 72hr time period, IC 50 values were not obtained.
  • TSA concentration is reduced by titrating to levels (10-50 nM) that should maintain maximal HDAC inhibition and minimal cytotoxicity.
  • HDAC1, 2, 3, and 6, among others, are potential candidate enzymes capable of prodrug activation and they are all known to be TSA sensitive (Bradner, J.E. et al . 2010), hence this range of TSA concentration should sufficiently inhibit all of these enzymes. Should it prove impossible to find a range of TSA that allows 72 hr cell toxicity assays, the use of other HDAC inhibitors is explored by routine experimentation. For example, among available HDAC inhibitors, SAHA (Vorinostat) , a FDA approved pharmaceutical HDAC inhibitor (Bradner, J.E. et al . 2010), is known to inhibit all of these HDACs with much lower cytotoxicity than TSA. Thus, SAHA is an alternative HDAC inhibitor for the prodrug evaluation as well. By comparing the prodrug IC 50 values for cytotoxicity under conditions of HDAC inhibition with those values where HDACs are active we are able to provide data on the HDAC dependence of our new class of prodrugs .
  • Boc-KAc-Puro a novel prodrug targeting increased HDAC and CTSL activities in malignant tumors.
  • Anticancer efficacy of the agent is evidenced by its ability to inhibit tumor growth in vivo without severe adverse effects.
  • Targeting elevated HDAC and CTSL activities in malignant cancer cells has been established as a strategy for anticancer drug development.
  • this approach is advantageous because the simple small molecule masking group could be readily applied to many other cytotoxic agents to confer selectivity that substantially improves their therapeutic index.
  • a potent and selective anticancer agent has been developed from a mere general cytotoxic drug Puro.
  • the strategy disclose herein is applied to many chemotherapeutic drugs currently used in the clinics aiming to improve their anticancer efficacy and safety.
  • amide coupling-based prodrugs of gemcitabine and cytarabine were developed by masking their amino group (Bender, D.M. et al. 2009; Cheon, E.P. et al. 2006) .
  • Boc-Lys (Ac) group is feasible for evaluation of their improved efficacy.
  • malignant cancer cells exhibit high levels of HDAC and CTSL activities has important implications for the potential use of their combined enzymatic activities as a selective modality for delivering therapeutics to their targets.
  • a prototypic agent was specifically activated by these enzymes in cancer cells. Such agents have the potential to improve clinical outcomes as well as quality of life for the patients.
  • HDAC histone deacetylase
  • CTSL endogenous protease cathepsin L
  • Goulet, B., et al A cathepsin L isoform that is devoid of a signal peptide localizes to the nucleus in S phase and processes the CDP/Cux transcription factor.
  • Molecular cell 14, 207-219 (2004) Goulet, B., et al .
  • Increased expression and activity of nuclear cathepsin L in cancer cells suggests a novel mechanism of cell transformation.
  • Molecular cancer research MCR 5, 899-907 (2007) .
  • XXI Effect of antibiotics on peptidyl-puromycin synthesis by mammalian polyribosomes. J Biol Chem 247, 6895-6900 (1972) . Richard, J. et al . Latent Fluorophores Based on a Self-Immolative Linker Strategy and Suitable for Protease Sensing. Bioconjugate Chem 19, 1707-1718 (2008) . Schmidt, E.K., Clavarino, G. , Ceppi, M. & Pierre, P. SUnSET, a nonradioactive method to monitor protein synthesis. Nature methods 6, 275-277 (2009) .
  • Vecsey-Sem en, B., et al Novel colon cancer cell lines leading to better understanding of the diversity of respective primary cancers.

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Abstract

La présente invention concerne un composé ayant la structure : dans laquelle X représente un agent thérapeutique contenant au moins un azote d'amine et l'azote d'amine sur l'agent thérapeutique se lie de façon covalente directement au carbone α ; Z représente CH3 ou CF3 ; R1 représente -H, -NR2R3, -NH-C (=O) -R4, -NH-C (=O) -OR4, -CH2-C (=O) -NR5R6, -OR7, -CO2R7, alkyle en C1-6, alcényle en C2-6, alcynyle en C2-6, aryle ou hétéroaryle, R2, R3, R4, R5, R6 et R7 représentant chacun indépendamment -H, alkyle en C1-6, alcényle en C2-6, alcynyle en C2-6, hétéroalkyle, cycloalkyle, hétérocyclyle, aryle, alkylaryle, hétéroaryle, alkylhétéroaryle, un acide aminé ou un oligopeptide ; une amine de l'acide aminé ou de l'oligopeptide étant substituée ou non substituée ; et n est un entier de 0 à 6 ; ou un diastéréisomère, un énantiomère ou un sel pharmaceutiquement acceptable du composé.
PCT/US2013/060443 2012-09-19 2013-09-18 Nouveaux promédicaments pour une thérapie anticancéreuse sélective WO2014047199A1 (fr)

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US10322192B2 (en) 2016-03-02 2019-06-18 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
US10722527B2 (en) 2015-04-10 2020-07-28 Capsugel Belgium Nv Abiraterone acetate lipid formulations
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US10435429B2 (en) 2017-10-03 2019-10-08 Nucorion Pharmaceuticals, Inc. 5-fluorouridine monophosphate cyclic triester compounds
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US10973920B2 (en) 2014-06-30 2021-04-13 Glykos Finland Oy Saccharide derivative of a toxic payload and antibody conjugates thereof
US10722527B2 (en) 2015-04-10 2020-07-28 Capsugel Belgium Nv Abiraterone acetate lipid formulations
US10322192B2 (en) 2016-03-02 2019-06-18 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
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